PROJECT SUMMARY: The mechanism of mycobacterial protein secretion remains elusive. The long-term goal of the applicant is to understand the molecular mechanisms of mycobacterial protein secretion. The overall objective of this applica- tion is to rigorously determine if the outer lipid, PDIM (phthiocerol dimycocerosate), is required for protein secre- tion. The central hypothesis is that PDIM promotes the optimal secretion of mycobacterial proteins across the MOM (mycobacterial outer membrane). The rationale underlying this proposal is that the successful completion of the proposed work will potentially inform the elusive mechanisms of protein secretion across the envelope while resolving an open question in the field regarding the role of PDIM in protein secretion. The central hypoth- esis will be tested by following this specific aim: Define the requirement for PDIM biosynthesis and transport in protein secretion. Under this aim, a combination of genetics with targeted and global quantitative proteomics will be used to measure changes in protein secretion in the presence or absence of PDIM production and localiza- tion. The expected outcome following the successful completion of the proposed aim will be the definitive deter- mination of the requirement of PDIM in mycobacterial protein secretion. This contribution will be significant be- cause it will directly inform the elusive mechanism of mycobacterial protein secretion and potentially identify the ESX-1 substrates that promote membrane lysis, advancing the field vertically. Moreover, completion of this pro- posal will contribute fundamental knowledge that could positively impact the treatment and prevention of the human disease, tuberculosis. The proposed research is conceptually innovative because it focuses on protein secretion independent of a class secretory apparatus. Moreover, the proposal provides an innovative explanation which could unify two disparate studies in the literature regarding the role of PDIM in protein secretion. Defining the relationship of the outer lipid PDIM with secretion will advance the field vertically by providing new insight into the molecular mechanisms of mycobacterial protein secretion.